摘要
本试验主要探讨白头翁复方制剂对腹泻小鼠肠道粘膜双糖酶活性的影响,以揭示白头翁复方制剂治疗腹泻的作用机理。试验方法：通过腹腔注射大肠杆菌建立动物腹泻模型,将试验小鼠分为预防组、治疗组、自愈组和空白对照组,攻菌小鼠每只腹腔注射2.6亿大肠杆菌O101诱发腹泻。预防组：提前7d开始用白头翁复方提取物灌胃直到试验结束,每次灌服1mL,每天一次；治疗组：攻菌造模后开始用白头翁复方提取物灌胃,每天一次；自愈组：仅用大肠杆菌进行造模；空白对照组：白头翁复方制剂和大肠杆菌肉汤悬液均用生理盐水代替。每天观察小鼠的临床症状,每天每组剖检6只小鼠,观察记录剖检变化,取十二指肠后段固定,采用常规切片技术观察肠道病理组织学变化,以探讨白头翁复方对小鼠腹泻的防治效果；参照葡萄糖氧化酶法,将肠道分为三段,刮取肠粘膜,匀浆后冷冻保存,测定各段肠粘膜的乳糖酶、麦芽糖酶和蔗糖酶的活性,进行比较分析,并用PCR方法对小鼠肠粘膜的乳糖酶基因的表达情况进行研究,以揭示白头翁复方治疗腹泻的作用机理。
试验结果如下：
临床症状：小鼠接种大肠杆菌O101后,4h后开始出现精神沉郁、倦怠、寒战、腹泻症状,临床表现为肛门粘有黄色稀便,采食量下降,眼睛的分泌物增多,被毛逆立,出现扎堆现象。对照组小鼠采食量正常,精神状态良好,未见异常表现。给药后第3天预防组和治疗组小鼠腹泻症状减轻,肛门干净,采食量正常；自愈组到第6天腹泻症状减轻,小鼠消瘦,精神沉郁,采食量比对照组减少。
剖检变化：攻菌造模后,预防组、治疗组和自愈组小鼠的肝脏、肾脏和脾脏肿大,胃鼓气严重,肠道呈片状出血,十二指肠最为明显,肠系膜淋巴结肿胀,肠道有黄色稀便,肠道变薄易拉断；对照组肠道未见异常。治疗组和预防组在给药3天后剖检肠道无出血现象,肠道后段粪便呈粒状；自愈组在给药6天后肠道前段出血减少,淋巴结肿胀减轻。
病理组织学变化：小鼠接种细菌发生腹泻后,小肠绒毛断裂,绒毛上皮细胞肿胀,部分坏死脱落,粘膜下层充血水肿,有少量炎性细胞浸润,杯状细胞数量增多,肠腺变性明显。治疗组和预防组在第4天后,病变有所减轻并逐渐恢复；与自愈组相比治疗组杯状细胞数量变化不显著,病理变化逐渐减轻；预防组杯状细胞数量和粘膜下层充血水肿变化不显著,空白对照组肠道组织结构正常,绒毛排列整齐,肠腺轮廓清晰。
肠粘膜双糖酶的变化：
①乳糖酶活性变化：通过比较各段乳糖酶的活性结果发现,肠道前段和中段的乳糖酶活性显著高于后段。接种大肠杆菌后第1天,预防组、治疗组和自愈组小鼠肠道粘膜乳糖酶活性显著下降,与对照组相比差异极显著(P<0.01),自愈组、预防组和治疗组三组之间差异不显著(P>0.05)；试验第2天,预防组和治疗组乳糖酶活性显著高于自愈组,且差异极显著(P<0.01)；试验第6天,预防组和治疗组与对照组相比差异不显著,与自愈组相比差异极显著(P<0.01)；
②蔗糖酶活性变化：试验第1天,接种大肠杆菌后测得各组蔗糖酶活性均显著降低与对照组相比差异极显著(P<0.01),但自愈组、预防组和治疗组三组之间差异不显著(P>0.05),试验第2天,治疗组和预防组各段蔗糖酶活性显著高于自愈组,且差异极显著(P<0.01),试验第6天,预防组和治疗组与对照组相比差异不显著,与自愈组相比差异极显著(P<0.01)；
③麦芽糖酶活性变化：接种大肠杆菌后试验第1天测得,自愈组、预防组和治疗组三组麦芽糖酶活性差异不显著(P>0.05),与对照组相比活性显著降低,且差异极显著(P<0.01)；试验第7天测得,预防组和治疗组与对照组相比差异不显著(P>0.05),与自愈组相比差异极显著(P<0.01)。
粘膜乳糖酶基因表达检测结果显示：与对照组相比,试验第1天,自愈组、治疗组和预防组小鼠Lac基因的表达量减少,且差异极显著(P<0.01),治疗组和预防组与自愈组相比差异不显著(P>0.05)；试验第5天,治疗组和预防组与对照组相比,差异不显著(P>0.05),但与与自愈组相比差异显著(P<0.05)；
结论：通过比较各组小鼠在临床症状和剖检变化得出,白头翁复方对小鼠大肠杆菌性腹泻有显著的防治作用；通过比较各组双糖酶的数据结果得出,白头翁复方能够明显促进腹泻小鼠肠道双糖酶活性的恢复,且对受损肠道乳糖酶的活性有一定的预防作用,且能显著促进肠道乳糖酶基因表达,结果表明白头翁复方对小鼠大肠杆菌性腹泻的防治效果可能与调控肠道粘膜双糖酶活性密切相关。
In order to investigate the prevention and cure mechanism of compound radix pulsatillae decoction (RP) for diarrhea, this test focuses on the effects of RP on the activitie of disaccharidase in intestinal mucosa in diarrheal mice induced with E. Coli. Method:one hundred and eighty BALB/c mice were randomly assigned into four groups:Prevention group, Treatment group, Self-healing group and Control group. Each mice was injected intraperitoneal with 2.6 billion E.coli O101 to induce diarrhea in all groups except the control group.The mice of Prevention group were fed with 1 mL RP once daily 7 days before the infection and last till the end of the test; The mice of Treatment group were fed with 1 mL RP once a day post infection;The mice of Self-healing group were fed with 1 mL physiological saline as the substitute once a day post infection; The mice of control group were treated with physiological saline as the substitute. The clinical symptoms were observed everyday.6 mice of each group were randomly selected for dissection on the every days post infection, the pathologic changes were observed. Posterior segment of duodenum was fixed immediately for making paraffin section. Histopathology changes were observed by light microscope. The mucosa of duodenum, jejunum and ileum were scraped, mashed and refrigerated respectively. The activities of lactase, maltase and sucrase of the mucosa homogenate were measured by the glucose oxidase method.The expression of the lactase gene of intestinal mucosa were measured by PCR method.
The results as follow:
Clinical symptoms:4h post infection the mice appeared depression, fatigue, chill, diarrhea and less feed intake.3d post infection the symptoms of prevention group and treatment group were decreased, the diarrhea were stopped. The symptoms of self-healing group were gradually decreased until 6d post infection. The mice of control group showed normally.
Pathologic changes:the mice of prevention group, treatment group, and self-healing group appeared flatulence and hemorrhage in intestine severely, the liver, kidney, spleen enlarged, mesenteric lymph node were swelling, and intestine is full of yellow Loose stools, and intestinal tract was thinner to pull off. The mice of control group showed normally.3d post infection the symptoms of prevention group and treatment group were no hemorrhage, stool was grainy in rectum.
Histopathological changes:the mice of prevention group,treatment group, and self-healing group appeared diarrhea after infected E. coli, the small intestine villi appeared partial ruptured, villous epithelial cell were swelled and necrosis partly, submucosa were congestion, edema and some inflammatory cell infiltration.The numbers of goblet cells were increased. The intestinal gland became degeneration obviously.4d post infection the symptoms of prevention group and treatment group were gradually decreased. Compared with the self-healing, the number of goblet cells did not increased significantly in treatment group, and other lesions consistent with the self-healing group. The number of goblet cells and submucosal edema had no significant changes in prevention group, Control group mice had the normal intestinal organizational structure.
Intestinal mucosa disaccharidase changes:
①Changes of lactase activity:it shows that duodenum and jejunum lactase activity is much higher than ileum after infected E. coli.The lactase activity of prevention group, treatment group and self-healing group were significantly lower than that of control group(P<0.01). The lactase activity of self-healing group, prevention group and treatment group had no differences one day post the infection (P>0.05).2d past infection the lactase activity of prevention and treatment groups was significantly higher than that of the self-healing group(P<0.01); 6d post infection, the lactase activity of prevention group, treatment group and control group were no significant difference but they were all significantly higher than that of the self-healing group(P<0.01);
②Changes of invertase activity:The invertase activity of prevention group, treatment group and self-healing group were significantly lower than that of control group (P<0.01). The invertase activity of self-healing group, prevention group and treatment group had no differences one day post the infection (P>0.05).2d past infection the invertase activity of prevention and treatment groups was significantly higher than that of the self-healing group(P<0.01); 6d post infection, the invertase activity of prevention group, treatment group and control group were no significant difference but they were all significantly higher than that of the self-healing group(P＜0.01);
③Changes of maltase activity:The maltase activity of prevention group, treatment group and self-healing group were significantly lower than that of control group(P<0.01), and they had no differences one day post the infection(P>0.05).7d post infection, the maltase activity of prevention group, treatment group and control group were no significant difference but they were all significantly higher than that of the self-healing group(P<0.01);
The expression of mucosal lactase gene display:The expression of Gene Lac of prevention group, treatment group and self-healing group were significant lower than that of control group (P<0.01). The expression of Gene Lac of self-healing group, prevention group and treatment group had no differences one day post the infection (P>0.05). (P>0.05); 5d post infection,the expression of Gene Lac of prevention group, treatment group and control group were no significant difference but they were all significantly higher than that of the self-healing group(P<0.01);
Conclusion:According to clinical symptoms,pathologic changes, histopathology changes and the activity of disaccharidase of the mice in each group, compound radix pulsatilla have significant preventive effects for diarrhea in mice, it can promote activity recovery of intestinal disaccharidase and the gene expression of intestinal lactase. The results showed that compound radix pulsatilla had the significantly preventive effects for diarrheal mice, and the preventive effect of compound radix pulsatilla may be closely related to regulat the activity of mucosal disaccharidase in intestinal.
引文
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